Flow cytometry is a technique for counting, examining and sorting microscopic particles suspended in a stream of fluid. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of single cells flowing through an optical/electronic detection apparatus. A beam of light, usually laser light, of a single frequency (color) is directed onto a hydrodynamically focused stream of fluid. A number of detectors are aimed at the point where the stream passes through the light beam; one in line with the light beam (Forward Scatter or FSC) and several perpendicular to it (Side Scatter or SSC) and one or more fluorescent detectors. Each suspended particle passing through the beam scatters the light in some way, and fluorescent chemicals in the particle may be excited into emitting light at a lower frequency than the light source. This combination of scattered and fluorescent light is picked up by the detectors, and by analyzing fluctuations in brightness at each detector (one for each fluorescent emission peak), it is possible to deduce various facts about the physical and chemical structure of each individual particle. FSC correlates with the cell size and SSC depends on the inner complexity of the particle, such as shape of the nucleus, the amount and type of cytoplasmic granules or the membrane roughness.
Flow cytometry systems or flow cytometers convert light signals from a cell sample or particle to electronic pulses and then an analog-to-digital converter (ADC) coverts the electronic pulses to channel numbers. The channel numbers represent flow cytometry data which can be presented on a display, such as a computer screen or printer, with the maximum acquired channel being presented at the end of the display object. As the technology for acquisition modules improves, cytometers are collecting wider dynamic data channel ranges. For example, instead of collecting four decades of dynamic data range, cytometers can now collect six decades of dynamic data range. Under this broader data range, data previously viewed in channels for three or four decades of dynamic data range are now viewed in six decades of dynamic data range.
Known flow cytometry systems present data on displays with the maximum acquired channel being presented at the end of the display object. For example older cytometers that only collected data from channel ranges 0 to 1023, would display the data on the display object from channels 0 to 1023. Newer cytometers, which collect broader data ranges (e.g., example 0 to 262,144), adjust the end of the display to 262,144 to accommodate the broader data ranges collected. In other words, prior art flow cytometer systems present the collected flow cytometry data with a fixed scale, and adjusts the flow cytometer hardware settings such as hardware gain, light source intensity, or PMT voltage in order to move particles with varying intensities to different locations in the fixed display area. A disadvantage of these systems is that in order to present the flow cytometry data at a desired location in the fixed area display, the cell sample must be rerun through the flow cytometer, at the adjusted hardware settings. These systems may be imprecise and the cell sample may have to be rerun several times before finding the desired settings of gain, intensity or voltage. In addition, old cell samples are not always available to be rerun and valuable data can be lost.
Because current flow cytometry systems fix the end of the channel range to the end of the display, in order to view cell samples having particles with varying intensities in a different location in the fixed display area, adjustments must be made to the flow cytometer hardware settings (e.g., hardware gain, light source intensity, and PMT voltage) and the cell sample must be rerun through the flow cytometer.
Thus, there exists a need for a more efficient cytometry system which can present cytometry data in alternate locations on the fixed area display object without the need to adjust cytometer instrument hardware settings and rerun the cell sample through the flow cytometer. The present invention satisfies this need and provides related advantages as well.
The present system and method, described herein, allows for a fixed hardware setting for all the possible range of particle sizes and intensities. Instead of adjusting the hardware settings, a mathematical operator or software gain is applied to the flow cytometry data. The data can then be presented in a different location on the display object other than the original fixed gain location.